Mefloquine synergism with anti-tuberculosis drugs and correlation to membrane effects: Biologic, spectroscopic and molecular dynamics simulations studies.

Studies displaying the combination of mefloquine (MFL) with anti-tuberculosis (TB) substances are limited in the literature. In this work, the effect of MFL-association with two first-line anti-TB drugs and six fluoroquinolones was evaluated against Mycobacterium tuberculosis drug resistant strains. MFL showed synergistic interaction with isoniazid, pyrazinamide, and several fluoroquinolones, reaching fractional inhibitory concentration indexes (FICIs) ranging from 0.03 to 0.5. In order to better understand the observed results, two approaches have been explored: (i) spectroscopic responses attributed to the effect of MFL on physicochemical properties related to a liposomal membrane model composed by soybean asolectin; (ii) molecular dynamics (MD) simulation data regarding MFL interaction with a membrane model based on PIM2, a lipid constituent of the mycobacterial cell wall. FTIR and NMR data showed that MFL affects expressively the region between the phosphate and the first methylene groups of soybean asolectin membranes, disordering these regions. MD simulations results detected high MFL density in the glycolipid interface and showed that the drug increases the membrane lateral diffusion, enhancing its permeability. The obtained results suggest that synergistic activities related to MFL are attributed to its effect of lipid disorder and membrane permeability enhancement.

In Vitro Anti/Pro-oxidant Activities of R. ferruginea Extract and Its Effect on Glioma Cell Viability: Correlation with Phenolic Compound Content and Effects on Membrane Dynamics.

Rapanea ferruginea antioxidant and antitumoral properties were not explored before in literature. This study aimed to investigate these biological activities for the R. ferruginea leaf extract and correlate them with its phenolic content and influence in biological membrane dynamics. Thus, in this study, anti/pro-oxidative properties of R. ferruginea leaf extract by in vitro DPPH and TBARS assays, with respect to the free radical reducing potential and to its activity regarding membrane free radical-induced peroxidation, respectively. Furthermore, preliminary tests related to the extract effect on in vitro glioma cell viability were also performed. In parallel, the phenolic content was detected by HPLC-DAD and included syringic and trans-cinnamic acids, quercetrin, catechin, quercetin, and gallic acid. In an attempt to correlate the biological activity of R. ferruginea extract and its effect on membrane dynamics, the molecular interaction between the extract and a liposomal model with natural-sourced phospholipids was investigated. Location and changes in vibrational, rotational, and translational lipid motions, as well as in the phase state of liposomes, induced by R. ferruginea extract, were monitored by Fourier-transform infrared spectroscopy, nuclear magnetic resonance, differential scanning calorimetry, and UV-visible spectroscopy. In its free form, the extract showed promising in vitro antioxidant properties. Free-form extract (at 1000µ g/mL) exposure reduced glioma cell in vitro viability in 40%, as evidenced by MTT tests. Pro-oxidant behavior was observed when the extract was loaded into liposomes. A 70.8% cell viability reduction was achieved with 500 µg/mL of liposome-loaded extract. The compounds of R. ferruginea extract ordered liposome interface and disorder edits a polar region. Phenolic content, as well as membrane interaction and modulation may have an important role in the oxidative and antitumoral activities of the R. ferruginea leaf extract.

Lipid-based nanocarrier for quercetin delivery: system characterization and molecular interactions studies.

The flavonoid quercetin (QU) is a naturally occurring compound with several biological activities. However, the oral bioavailability of this compound is very low due to the high pre-systemic metabolism in the colon and liver and its low water solubility. In this context, the development of QU-loaded nanocarriers (NEs) is a promising approach to improve the drug oral bioavailability. This study investigates the variation of the concentration of 12-hydroxystearic acid-polyethylene glycol copolymer, lecithin and castor oil (CO) as to increase the amount of QU encapsulated while maintaining physicochemical characteristics described in previous studies. To better understand the ability to load and release the drug, we investigated the molecular interactions between QU and NE. Lipid-based NEs were prepared using CO as oily phase and PEG 660-stearate and lecithin as surfactants. Hot solvent diffusion and phase inversion temperature were methods employed to produce NEs. The QU-NEs were investigated for physicochemical characteristics and in vitro drug release. Molecular interactions between QU and the NEs were monitored through the complementary infrared (Fourier transform infrared) and NMR. The results revealed that it was possible to incorporate higher amounts of QU in a lipid-based NE with a reduced size (20 nm). The system developed allow a sustained release of QU probably due to the shell formed by the surfactants around the NE and the flavonoid ordering effect in the emulsion hydrophobic regions, which may reduce the system permeability.

Benzohydroxamate and nitrobenzohydroxamate affect membrane order: Correlations between spectroscopic and molecular dynamics to approach tuberculosis.

This work correlates the effects of benzohydroxamate (BH) and nitrobenzohydroxamate (NBH) anions in two membrane models which may be used for anti-tuberculosis (anti-TB) spectroscopic studies and/or computational studies. Firstly, the BH and NBH influence in the physico-chemical properties of soy asolectin (ASO)-based large multilamellar vesicles (MLVs) were evaluated by spectroscopic and calorimetric studies. In parallel, the BH and NBH interaction with a Mycobacterium tuberculosis (Mtb) inner membrane model, composed of phosphatidyl-myo-inositol-dimannoside (PIM2), was investigated by molecular dynamics (MD) simulations. Spectroscopic data showed a localization of BH close to the lipid phosphate group, while NBH was found close to the choline region. The BH ordered the ASO choline, phosphate and carbonyl regions and disrupted the acyl methylenes, reducing the membrane packing of the lipid hydrophobic region. On the other hand, NBH showed an ordering effect in all the lipid groups (polar, interface and hydrophobic ones). By MD studies, it was found that NBH enhanced the stability of the PIM2 membrane more than BH, while also being positioned closer to its mannosyl oxygens. As in ASO MLVs, BH was localized close to the PIM2 phosphate group and disrupted its acyl chains. However, higher values of lateral diffusion were observed for NBH than BH. Despite this, BH and NBH increased the membrane thickness by 35 %, which suggests a global ordering effect of both drugs. Findings of this work reinforce the accordance and complementarity between MLVs based on ASO and the PIM2 MD model results to study the drug effects in Mtb membrane properties.

Effects of α-eleostearic acid on asolectin liposomes dynamics: relevance to its antioxidant activity.

In this study, the effect of α-eleostearic acid (α-ESA) on the lipid peroxidation of soybean asolectin (ASO) liposomes was investigated. This effect was correlated to changes caused by the fatty acid in the membrane dynamics. The influence of α-ESA on the dynamic properties of liposomes, such as hydration, mobility and order, were followed by horizontal attenuated total reflection Fourier transform infrared spectroscopy (HATR-FTIR), nuclear magnetic resonance (NMR), differential scanning calorimetry (DSC) and UV-vis techniques. The α-ESA showed an in vitro antioxidant activity against the damage induced by hydroxyl radical (OH) in ASO liposomes. The analysis of HATR-FTIR frequency shifts and bandwidths and (1)H NMR spin-lattice relaxation times, related to specific lipid groups, showed that α-ESA causes an ordering effect on the polar and interfacial regions of ASO liposomes, which may restrict the OH diffusion in the membrane. The DSC enthalpy variation analysis suggested that the fatty acid promoted a disordering effect on lipid hydrophobic regions, which may facilitate interactions between the reactive specie and α-ESA. Turbidity results showed that α-ESA induces a global disordering effect on ASO liposomes, which may be attributed to a change in the lipid geometry and shape. Results of this study may allow a more complete view of α-ESA antioxidant mode of action against OH, considering its influence on the membrane dynamics.

Structure and interaction roles in the release profile of chalcone-loaded liposomes.

The structures and molecular interactions of established synthetic chalcones were correlated with their release profiles from asolectin liposomes. The effects of chalcones on the properties of liposomes were evaluated by dynamic light scattering (DLS), ultraviolet-visible spectroscopy (UV-VIS), horizontal attenuated total reflection Fourier transform infrared (HATR-FTIR), 31P nuclear magnetic resonance (31P NMR), zeta (ζ) potential and differential scanning calorimetry (DSC). The profiles and mechanisms of release were accessed according to the Korsmeyer-Peppas model. Results obtained allowed the establishment of a relationship between the chalcone release profile and 1) the ordering effects of chalcones in different membrane regions, 2) their polar or interfacial location in the lipid layer, 3) the influence of hydroxy and methoxy substituents, 4) their effect on reorientation of lipid choline-phosphate regions. The obtained data may improve the development of chalcone-based systems to be used in the therapy of chronic and acute diseases.

Spirulina sp. LEB 18-extracted phycocyanin: Effects on liposomes' physicochemical parameters and correlation with antiradical/antioxidant properties.

This study describes the physicochemical properties of soybean asolectin (ASO) liposomes loaded with phycocyanin (Phy) extracted from Spirulina sp. LEB 18. The effects of Phy in the liposomes' properties were investigated by Fourier transform infrared spectroscopy (FTIR), 1H and 31P nuclear magnetic resonance (NMR), zeta (ζ)-potential, dynamic light scattering (DLS) and ultraviolet-visible (UV-vis) techniques. Phy restricted the motion of ASO polar and interface groups and disrupted the package arrangement of the lipid hydrophobic regions, as a likely effect of dipolar and π interactions related to its amino acid residues and pyrrole portions. These interactions were correlated to antiradical/antioxidant Phy responses obtained by 2,2-diphenyl-1-picrylhidrazil (DPPH) assay, thiobarbituric acid reactive substances (TBARS) and ferric reducing antioxidant power (FRAP) methods, and discussed to bring new chemical perspectives about Phy-loaded liposomes-related nutraceutical applications in inflammatory and viral infection processes.

Physico-chemical interactions of a new rod-coil-rod polymer with liposomal system: Approaches to applications in tryptophan-related therapies.

This work describes the synthesis of the new supramolecular rod-coil-rod polymer, designated as cholesterol-PEO1000-tryptophan (Chl-PEO-Trp), as well as its effects on the physico-chemical properties of phosphatidylcholine (PC)-based liposomes. The molecular interactions between the Chl-PEO-Trp and PC were characterized by HATR-FTIR, DSC, NMR, DLS and zeta (ζ) potential techniques. The Chl-PEO-Trp polymer yield was 75 %. FTIR and DSC data showed that the motion of almost all PC groups was restricted by the polymer, and it promoted a decrease of the trans-gauche isomerization of the PC methylene, restricting the mobility of the hydrophobic region of the liposomes. NMR analyses indicated a Chl-PEO-Trp-induced restriction in the rotation of the PC phosphorus and a discreet increase of the hydrogen mobility of the choline. Despite this increase in the rotation of the choline, DLS and ζ-potential analyses suggested a reorientation of the choline group toward the system surface, which contributed, along with the other physico-chemical effects, to a globally packed membrane arrangement and reduced liposome size. Data described in this work were correlated to possible applications of the Chl-PEO-Trp in its free or PC liposome-loaded forms in the diagnosis and therapy of cancer, SARS caused by coronaviruses, and central nervous system-related diseases.

Dimiristoylphosphatidylcholine/genistein molecular interactions: A physico-chemical approach to anti-glioma drug delivery systems.

Regarding free genistein small delivery to the central nervous system, physico-chemical parameters of dimiristoylphosphatidylcholine liposome-loaded genistein were investigated, as well as its in vitro activity against the DPPH radical and glioma cells. Data obtained by UV-vis spectroscopy, Fourier Transform Infrared Spectroscopy, Nuclear Magnetic Resonance, Differential Scanning Calorimetry and Dynamic Light Scattering were used to characterize the liposomal system with respect to motion restriction, hydration degree, trans-gauche isomerization and phase state. In vitro antitumoral effects were monitored through conting and viability assays. Genistein hydroxyl group and lipid hydrogen bonds may have important role in dimiristoylphosphatidylcholine phosphate and choline motion restriction. Genistein-induced choline restriction may be also related to a decrease in the group rotation rate. Genistein: dimiristoylphosphatidylcholine system showed higher molecular package at the acyl chains region compaired to empty liposomes, and it may be related to a decrease in gauche bonds quantity and system size. Lipid acyl chain length seems to influence different genistein effects on membranes, due to the presence of gauche conformers. Genistein: dimiristoylphosphatidylcholine liposome was more efficient as DPPH reducting system than the free-Gen. Liposomal system, at genistein 100 µM, was so efficient as the correspondent free-form genistein, probably showing higher stability to cross the blood-brain barrier. Genistein and the lipid did not show an additive activity against glioma cells. Antioxidant and anti-glioma genistein-loaded liposome potential may be related to the isoflavone location and its restriction effect in the lipid molecular motion. Anti-glioma activity may also be related to a decrease of system size and trans-gauche isomerization.

Elucidating Bauhinia variegata lectin/phosphatidylcholine interactions in lectin-containing liposomes.

Investigations focused on the interactions of nanoparticles with lectins are relevant since it is well accepted that such proteins can be recognized by carbohydrates as parts of cell membranes. This can ultimately enhance the cellular uptake of the produced assemblies. In this framework, the physical interactions of phosphatidylcholine (PC) liposomes and the Bauhinia variegate lectin (BVL) are reported here. BVL-liposome interactions were characterized by a variety of techniques to understand the influence of BVL in the structural features, thermodynamic and spectroscopic properties of the hybrid material. The produced system is composed of 56% w/w lectin, and the scattering techniques show the presence of stable vesicular structures with a mean diameter DH ∼ 100 nm. The FTIR and NMR results showed a strong lectin effect on the PC choline region, restricting the rotational motion of the lipid group. The BVL-liposome interaction promoted hardening of the protein as evidenced by circular dichroism spectroscopy. The photophysics results suggest higher rigidity of the system in the presence of BVL. The BVL may be present in the inner or outer polar surface of the liposomes. The system was shown to be relatively stable and therefore potentially useful for carbohydrate recognition of nanoparticles.

Assessment of the encapsulation effect of phenolic compounds from Spirulina sp. LEB-18 on their antifusarium activities.

This study aimed to investigate the antifungal activity of liposomal systems containing Spirulina sp. LEB-18 phenolic extract (PE) against Fusarium graminearum (Fg) isolates. The interaction between PE and phosphatidylcholine-based liposomes was monitored by HATR-FTIR, NMR, DSC, and cryo-TEM. After encapsulation, the active principle was released slower than the free PE, a fact that makes the former very promising as a natural antifungal. The PE encapsulation in the liposomes was responsible for changes in the dynamics of specific regions. These compounds affected the membrane hydration degree, ordered the lipid phosphate region and increased the disorder of the acyl chain methylenes. These physico-chemical effects may be related to the strong inhibition of four Fg isolates. Results were discussed by correlating structural similarities, as well as the membrane effects of the PE under study on antifusarium activities, and those found in the literature, thus enabling the PE mechanisms of action to be analyzed.

Self-assembled carbohydrate-based vesicles for lectin targeting.

This study examined the physicochemical interactions between vesicles formed by phosphatidylcholine (PC) and glycosylated polymeric amphiphile N-acetyl-ß-d-glucosaminyl-PEG900-docosanate (C22PEG900GlcNAc) conjugated with Bauhinia variegata lectin (BVL). Lectins are proteins or glycoproteins capable of binding glycosylated membrane components. Accordingly, the surface functionalization by such entities is considered a potential strategy for targeted drug delivery. We observed increased hydrodynamic radii (RH) of PC+C22PEG900GlcNAc vesicles in the presence of lectins, suggesting that this aggregation was due to the interaction between lectins and the vesicular glycosylated surfaces. Furthermore, changes in the zeta potential of the vesicles with increasing lectin concentrations implied that the vesicular glycosylated surfaces were recognized by the investigated lectin. The presence of carbohydrate residues on vesicle surfaces and the ability of the vesicles to establish specific interactions with BVL were further explored using atomic force microscopy (AFM) and small-angle X-ray scattering (SAXS) analysis. The results indicated that the thickness of the hydrophilic layer was to some extent influenced by the presence of lectins. The presence of lectins required a higher degree of polydispersity as indicated by the width parameter of the log-normal distribution of size, which also suggested more irregular structures. Reflectance Fourier transform infrared (HATR-FTIR), differential scanning calorimetry (DSC), nuclear magnetic resonance (NMR) and ultraviolet-visible (UV-vis.) analyses revealed that the studied lectin preferentially interacted with the choline and carbonyl groups of the lipid, thereby changing the choline orientation and intermolecular interactions. The protein also discretely reduced the intermolecular communication of the hydrophobic acyl chains, resulting in a disordered state.

Physico-chemical characterization of asolectin-genistein liposomal system: An approach to analyze its in vitro antioxidant potential and effect in glioma cells viability.

In this study, the interaction between soy isoflavone genistein and asolectin liposomes was investigated by monitoring the effects of isoflavone on lipidic hydration, mobility, location and order. These properties were analyzed by the following techniques: horizontal attenuated total reflection Fourier transform infrared spectroscopy (HATR-FTIR), low-field (1)H nuclear magnetic resonance (NMR), high-field (31)P NMR, zeta potential, differential scanning calorimetry (DSC) and UV-vis spectroscopy. The antioxidant and antitumoral activities of the genistein liposomal system were also studied. The genistein saturation concentration in ASO liposomes corresponded to 484 µM. HATR-FTIR results indicated that genistein influences the dynamics of the lipidic phosphate, choline, carbonyl and acyl chain methylenes groups. At the lipid polar head, HATR-FTIR and (31)P NMR results showed that the isoflavone reduces the hydration degree of the phosphate group, as well as its mobility. Genistein ordered the lipid interfacial carbonyl group, as evidenced by the HATR-FTIR bandwidth analysis. This ordering effect was also observed in the lipidic hydrophobic region, by HATR-FTIR, NMR, DSC and turbidity responses. At the saturation concentration, liposome-loaded genistein inhibits the lipid peroxidation induced by hydroxyl radical in 90.9%. ASO liposome-loaded genistein at 100 µM decreased C6 glioma cell viability by 57% after 72 h of treatment. Results showed an increase of the genistein in vitro activities after its incorporation in liposomes. The data described in this work will contribute to a better understanding of the interaction between genistein and a natural-source membrane and of its influence on isoflavone biological activities. Furthermore, the antitumoral results showed that genistein-based liposomes, which contain natural-sourced lipids, may be promising as a drug delivery system to be used in the glioma therapy.

Relationship between the action of reactive oxygen and nitrogen species on bilayer membranes and antioxidants.

Membrane lipid peroxidation (LPO) induced by hydroxyl (*OH) and ascorbyl (*Asc) radicals and by peroxynitrite (ONOO-) was investigated in asolectin (ASO), egg phosphatidylcholine (PC) and PC/phosphatidic acid mixtures (PC:PA) liposomes and rat liver microsomes (MC). Enthalpy variation (DeltaH) of PC:PA at different molar ratios were obtained by differential scanning calorimetry. It was also evaluated the LPO inhibition by quercetin, melatonin and Vitamin B6. The oxidant effect power follows the order *OH approximately *Asc > ONOO- on PC and MC; whilst on ASO liposomes, it follows *Asc > *OH approximately ONOO-. Increasing amounts of PA in PC liposomes resulted in lower levels of LPO. The DeltaH values indicate a more ordered membrane arrangement as a function of PA amount. The results were discussed in order to provide a complete view involving the influence of membranes, oxidants and antioxidants intrinsic behavior on the LPO dynamics.

Melatonin location in egg phosphatidylcholine liposomes: possible relation to its antioxidant mechanisms.

Although it is known that the antioxidant properties of melatonin can be modulated by its effect on membrane fluidity, there are few studies on this subject reported in the literature and they are controversial. In this study, viscosimetry and nuclear magnetic resonance (NMR) techniques were used to determine melatonin's effect and location on egg phosphatidylcholine bilayers mobility. Melatonin decreases the dynamic viscosity of the lipid dispersion. (31)P-NMR line width analysis indicated that melatonin induces a slight but uniform restriction of the lipid motional freedom in the polar head. However, melatonin changes in choline (13)C dynamics was only observed through chemical shift analysis. On the other hand, melatonin can induce an increase in the lipid nonpolar chain mobility, as observed by (13)C and (1)H relaxation time analysis. These results suggest the interfacial location of melatonin in the membrane. Additionally, the results of the analysis of the lipid (1)H-fitted exponential relaxation times suggest that melatonin promotes a molecular rearrangement of the bilayers. The melatonin effect and location in the lipid membrane may explain its antioxidant properties against lipid peroxidation induced by reactive species.